This is a new proposal to understand the structure, function, regulation and small-molecule stimulation of the nitric oxide receptor, soluble guanylyl/guanylate cyclase (sGC). Nitric oxide (NO) is remarkable in that it is a small, toxic compound, but is produced by most cells to regulate diverse physiology, including blood pressure, wound healing, tissue development and memory formation. Loss of NO signaling leads to cardiovascular disease and is linked to the vascular disorders associated with diabetes, aging and related health failures. Promising new compounds targeting sGC are in clinical trial or in the clinic but how they function or even where they bind in many cases is unknown, making advancement challenging. sGC is a 150 kDa heterodimeric hemoprotein that responds to multiple factors through allosteric regulation, including stimulation by NO and by drug candidates. We have developed human and hawkmoth (Manduca sexta) sGC for addressing mechanism in NO signaling. We seek to understand, at a molecular level, how sGC functions in normal physiology, how it fails in cardiovascular disease and how drug binding can overcome these failures. We propose three specific aims: Aim 1: Atomic structures of sGC and its functional complexes. A key limitation in the nitric oxide field is the absence of structure for th nitric oxide receptor. We have produced new crystals of truncated heterodimeric sGC in the presence or absence of NO and stimulator compounds. These crystals should allow for atomic structures to be determined for both the active and inactive states, providing key insight into sGC activation and revealing the molecular details for drug binding and drug action. Aim 2: Mechanism for signal transduction in sGC. Binding of NO or candidate drugs to sGC leads to an ~200 fold increase in catalytic activity, but how signal transduction from the heme domain to the cyclase domain takes place is unknown. We propose to uncover the underlying signal transduction mechanism for sGC activation using UV-visible and luminescence decay spectroscopic measurements, engineered lanthanide binding tags and mutagenesis. Aim 3: sGC regulation through posttranslational modification. sGC response to NO is regulated in multiple ways, but most importantly through phosphorylation. We propose to discover where sGC is phosphorylated in response to extracellular signals, how phosphorylation inhibits the protein, which signaling pathway is responsible and when it occurs in the cell. The primary technique to be employed is mass spectrometry. We have previously shown that YC-1 family drug candidates can overcome sGC inhibition by phosphorylation. We expect a more complete understanding of cellular sGC regulation will provide much needed drug-discovery opportunities.